I'm doing library-scale ligation and transformation, cloning a 300bp gene-assembly fragment (containing randomized stretches) into a 3.5kb vector, and since we're aiming for a diversity of 10 billion variants, efficiency is of the utmost importance.
Running my ligation product out on a gel, I get bands at 3.5kb (empty vector) and 3.8kb (vector plus insert), and from the relative intensities I see that 25-33% contains the insert. Around 95% of my transformants contain insert (checked by cPCR), so the empty vector I'm seeing in the ligation product is presumably not circularized.
I'm trying to figure out whether I should be elated that I'm getting a quarter to a third of the theoretical maximum, or concerned that 75% of my vector isn't being ligated. While ligase manufacturers show 95% re-ligation of digested vector I've been unable to find an example of a "real-world" ligation of PCR-generated insert and gel-extracted, phosphatased vector showing what the efficiency is.
Anyone have any thoughts on this? Should I be trouble-shooting in an attempt to maybe double my efficiency, or should I be happy with what I've got?
Details:
Insert and vector digested with XhoI and BamHI, heat-inactivated
Vector phosphatased, heat-inactivated, gel-extracted
Insert affinity-purified
100ng Vector + 34ng insert (1:4 molar ratio) in 20 microlitre reaction, 1U T4 DNA ligase (NEB)
Thanks!
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